Solid-Phase Synthesis and Purification of Protein-DNA Origami Nanostructures
Solid-Phase Synthesis and Purification of Protein-DNA Origami Nanostructures
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DOI：
10.1002/chem.201805506
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发表时间：
2019-03-07
影响因子：
4.3
通讯作者：
Niemeyer, Christof M.
中科院分区：
文献类型：
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作者：
Burgahn, Teresa;Garrecht, Ruben;Niemeyer, Christof M.
我们提出了一种简单的方法，用于蛋白质修饰的DNA折纸纳米结构（DONs）的合成和纯化。除了用于连接重组蛋白的配体之外，还带有可还原切割的生物素基团的DON与磁珠结合。用大量过量的蛋白质进行蛋白质固定以实现高连接产率。在从固体支持物裂解之后，获得纯样品溶液，其适合于占据模式的直接AFM分析。我们使用三种不同的正交连接方法，“基于卤素的寡核苷酸结合剂”（HOB），Halo标签的变体，“SpyTag/SpyCatcher”（ST/SC）系统和酶促“ybbR标签”偶联来证明该方法的实用性。我们发现ST/SC连接的效率令人惊讶地低，可能是由于静电排斥和空间位阻，而ybbR方法，尽管其三元性质，显示出良好的连接产率。我们的方法是特别有用的新的连接方法的发展和机械脆弱的DON的合成，目前的蛋白质模式的表面为基础的细胞测定。
We present a facile method for the combined synthesis and purification of protein-decorated DNA origami nanostructures (DONs). DONs bearing reductively cleavable biotin groups in addition to ligands for ligation of recombinant proteins are bound to magnetic beads. Protein immobilization is conducted with a large protein excess to achieve high ligation yields. Subsequent to cleavage from the solid support, pure sample solutions are obtained which are suitable for direct AFM analysis of occupation patterns. We demonstrate the method's utility using three different orthogonal ligation methods, the "halo-based oligonucleotide binder" (HOB), a variant of Halo-tag, the "SpyTag/SpyCatcher" (ST/SC) system, and the enzymatic "ybbR tag" coupling. We find surprisingly low efficiency for ST/SC ligation, presumably due to electrostatic repulsion and steric hindrance, whereas the ybbR method, despite its ternary nature, shows good ligation yields. Our method is particularly useful for the development of novel ligation methods and the synthesis of mechanically fragile DONs that present protein patterns for surface-based cell assays.